Protein secretion is one of the most important and complex physiological processes in growing cells. The main aim of this proposal is to analyze the molecular details of this process in vitro, i.e. protein translocation into bacterial membrane vesicles. In addition, we will examine further how proteins are excreted past a double membrane in certain Gram-negative bacteria. The elucidation of the mechanism of protein translocation is greatly facilitated by our newly developed, efficient system utilizing extracts, mRNA for alkaline phosphatase and OmpA protein, and inner membrane vesicles, all from E. coli. This system can separate the stage of translocation from that of translation, which makes the biochemical analysis of protein translocation much simpler. With this system we will pursue indications that protein translocation requires ATP as well as protonmotive force, and whether a complex of proteins that may be the bacterial equivalent of the eukaryotic signal recognition particle, and other cytoplasmic proteins, are involved. To identify membrane proteins involved in translocation we will use partial reconstitution, protease inactivation, cross-linking of precursors of secreted proteins to membrane proteins, specific antibodies to block function, and mutants defective in secretion. To study protein excretion past a double membrane in Gram-negative bacteria, we will examine the biosynthesis and excretion pathway of Pseudomonas aeruginosa hemolysin and Vibrio cholera toxin. Building on evidence that excretion of toxin to the exterior requires genes not present in E. coli cells, we will introduce additional genes from Pseudomonas into E. coli and will seek transformants that excrete toxin to the exterior instead of secreting it into the periplasm. If the results are positive, the required gene(s) and protein(s) will be identified. An understanding of mechanisms discovered in bacteria is likely to have implications for secretion of proteins by human cells, and should have important practical implications for the medical-industrial use of recombinant DNA in bacteria. Furthermore, the studies of microbial toxins are directly relevant to medical bacteriology.